PROJECT SUMMARY The human bacterial pathogen Streptococcus pyogenes (group A streptococcus, GAS) causes more than 700 million human infections annually worldwide. The proposed research seeks to discover, on a genome- wide scale, GAS genes required for, or contributing to fitness in the female genital tract, an infection site for which very little basic understanding is known. We will use a serotype M28 GAS strain because for reasons that aren?t understood, these organisms are non-randomly associated with female genital tract disease, including puerperal sepsis (childbed fever) and neonatal invasive infections. The pathogen genes and fundamental molecular pathogenesis processes contributing to two phases of genital tract interaction will be studied: (i) colonization and proliferation and (ii) persistence. This basic science knowledge deficit has severely limited the ability to create new clinical tools such as a successful GAS vaccine. Thus, the studies are designed to address this critical knowledge deficit. We will combine our very recent success in applying transposon-directed insertion site sequencing (TraDIS) technology to GAS with our 18 years of productive experience with cynomolgus macaque models of GAS infection, currently the gold standard experimental animal to study this human pathogen. We will use TraDIS to conduct an in vivo genome-wide screen that will systematically identify GAS genes required for colonization and proliferation, and persistence in the genital tract of female cynomolgus macaques. The goal of the proposed studies is to identify new molecular mechanisms used by GAS to enhance fitness in this anatomic niche. The results of our studies may ultimately help in formulating novel translational strategies to prevent or treat GAS infections. To achieve our goals, the following two specific aims are proposed: Specific Aim 1: Exploit TraDIS for genome-wide identification of GAS genes required for (i) colonization and proliferation, and (ii) persistence (i.e., fitness) in the genital tract of female cynomolgus macaques. Specific Aim 2: Use isogenic gene-knockout mutant strains to validate the importance of four selected candidate genes to GAS colonization and proliferation, and persistence identified by the genome-wide screen. The proposed line of research will exploit our innovative and successful application of TraDIS to GAS, including infections in cynomolgus macaques. It will be the first use of TraDIS in the genital tract of female non-human primates and the first study to investigate determinants of GAS colonization and proliferation, and persistence, on a mucosal surface using genome-wide transposon mutagenesis.